Advances in the analytical methodologies: Profiling steroids in familiar pathways-challenging dogmas

• Advances in adrenal steroid profiling in health and disease using GC– and LC–MS/MS.
• UPLC–MS/MS quantification of steroids in Ang II and K+ stimulated H295R cells.
• UPC2–MS/MS analyses of 11OH-androstenedione and 11OH-testosterone metabolites.
• UHPLC–MS/MS analyses of A4 and T C11-hydroxy and C11-keto metabolites in LNCaP cells.
• 11OHA4’s potential role in prostate cancer, sexual development and PCOS is identified.

The comprehensive evaluation of the adrenal steroidogenic pathway, given its complexity, requires methodology beyond the standard techniques currently employed. Advances in LC–MS/MS, coupled with in vitro cell models that produce all the steroid metabolites of the mineralo-, glucocorticoid and androgen arms, present a powerful approach for the comprehensive evaluation of adrenal steroidogenesis in response to compounds of interest including bioactives, drug treatments and endocrine disrupting compounds. UHPLC–MS/MS analysis of steroid panels in forskolin, angiotensin II and K+ stimulated H295R cells provides a snapshot of their effect on intermediates and end products of adrenal steroidogenesis. The impact of full steroid panel evaluations by LC– and GC–MS/MS extends to clinical profiling with the characterization of normal pediatric steroid reference ranges in sexual development and of disease-specific profiles improving diagnosis and sub classification. Comprehensive analyses of steroid profiles may potentially improve patient outcomes together with the application of treatments specifically suited to clinical subgroups. LC–MS/MS and GC–MS/MS applications in the analyses of comprehensive steroid panels are demonstrated in clinical conditions such as congenital adrenal hyperplasia in newborns requiring accurate diagnoses and in predicting metabolic risk in polycystic ovary syndrome patients. Most notable perhaps is the impact of LC–MS/MS evaluations on our understanding of the basic biochemistry of steroidogenesis with the detection of the long forgotten adrenal steroid, 11β-hydroxyandrostenedione, at significant levels. The characterization of its metabolism to androgen receptor ligands in the LNCaP prostate cancel cell model, specifically within the context of recurring prostate cancer, lends new perspectives to old dogmas. We demonstrate that UHPLC–MS/MS has enabled the analyses of novel metabolites of the enzymes, SRD5A, 11βHSD and 17βHSD, in LNCaP cells. Undoubtedly, the continuous advances in the analytical methodologies used for steroid profiling and quantification will give impetus to the unraveling of the remaining enigmas, old and new, of both hormone biosynthesis and metabolism.